番茄细菌性斑点病菌实时荧光定量PCR检测方法的建立及应用

Development and Application of Quantitative PCR for Detection of Pseudomonas syringae pv. tomato

以番茄细菌性斑点病病原菌丁香假单胞菌番茄致病变种(Pseudomonas syringae pv. tomato,Pst)的致病相关基因HrpZ为靶序列,设计了特异性引物Pst3F/Pst3R,能从Pst基因组DNA中特异性扩增出大小为161 bp的目的片段。建立的Pst实时荧光定量PCR检测技术体系的检测灵敏度比普通PCR高1 000倍。利用实时荧光定量PCR检测体系,检测模拟带菌种子中Pst的带菌量,检测下限为4.21 cfu·g^(-1);检测人工接种叶片组织中Pst的带菌量,检测到1级发病叶片带菌量为4.15×102 cfu·g^(-1)。对田间采集的63个番茄细菌性斑点病明显症状和疑似症状样本,分别进行了实时荧光定量PCR、普通PCR和病原菌分离检测,检测到54个样本中含有Pst,3种方法检测结果一致。结果表明,建立的Pst实时荧光定量PCR检测体系具有特异性强、灵敏度高的特点,可以快速准确地定量检测番茄种子和发病组织中Pst的含量,为番茄细菌性斑点病的早期预防和流行监测提供了有效的技术手段。

In the present work,a specific primer pair Pst3F/Pst3R was designed based on the pathogenicity-related gene HrpZ of Pseudomonas syringae pv. tomato(Pst),and a target fragment of 161 bp was specifically amplified from the genomic DNA of Pst. A real-time fluorescent quantitative PCR(qPCR)assay was developed for quantifying Pst in contaminated tomato seeds and infected tissues. The detection sensitivity of qPCR established in this study was 1 000 times higher than that of conventional PCR. The detection threshold was 4.21 cfu per gram of artificially contaminated tomato seeds. The amount of Pst was detected to be 4.15 × 10^2 cfu per gram of inoculated leaves with disease-grade level 1. For naturally infected tomato tissue samples,PCR,qPCR and classical culture methods were used for determination of Pst. Quantifiable levels of Pst were detected in 54 out of the 63 samples,and the results of the three methods were consistent. These studies showed that qPCR assay is a highly rapid and reliable method to quantify Pst in tomato seeds and infected tissues of leaves,stems and fruits. Application of the assay may potentially improve pathogen identification and disease management.